Journal of Gastroenterology

, Volume 46, Issue 12, pp 1391–1402 | Cite as

Differential expression of miR-144* as a novel fecal-based diagnostic marker for colorectal cancer

  • Murugan Kalimutho
  • Giovanna Del Vecchio Blanco
  • Serena Di Cecilia
  • Pierpaolo Sileri
  • Micaela Cretella
  • Francesco Pallone
  • Giorgio Federici
  • Sergio Bernardini
Original Article—Alimentary Tract



MicroRNAs (miRNA) are tiny, noncoding, small, endogenous RNAs that play major roles in neoplastic transformation and could therefore offer a better quantitative and noninvasive method for the diagnosis and prognosis of colorectal cancer (CRC) using feces. In the present study, we screened feces for 648 miRNAs and analyzed the role of miR-144* as a potential CRC diagnostic marker.


Fecal miRNA expression was profiled with RT-pre-amplification-qPCR, and the stability was determined using both endogenous and exogenous miRNA by RT-qPCR. ROC analysis was performed to enhance the diagnosing power of the CRC patients’ fecal specimens.


We detected 39% of all the miRNAs screened in feces. Endogenous miRNAs are more stable over time and temperature, while exogenous miRNAs degraded rapidly. miR-144* was overexpressed in feces, suggesting that it could be a potent candidate diagnostic marker for CRC detection, with a sensitivity of 74% and a specificity of 87% (n = 75, p < 0.0001). Moreover, RT-qPCR analysis showed that miR-144* was also overexpressed in paired CRC tissues, thus suggesting its possible utilization as a diagnostic marker.


We demonstrated that miRNAs are stable in the fecal microenvironment, and that, among them, miR-144* represents a novel fecal-based diagnostic marker for CRC screening. Nevertheless, our data need to be validated in a large cohort of subjects.


miR-144* Fecal miRNAs CRC miR-378 





Fecal DNA


Colorectal cancer



This work was supported by grants from University Hospital Tor Vergata, University of Rome. MK was supported by a pre-doctoral scholarship for foreign students under the International Italian Government University scholarship.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Cummins JM, He Y, Leary RJ, Pagliarini R, Diaz LA Jr, Sjoblom T, et al. The colorectal microRNAome. Proc Natl Acad Sci USA. 2006;103:3687–92.PubMedCrossRefGoogle Scholar
  2. 2.
    Akao Y, Nakagawa Y, Naoe T. MicroRNA-143 and -145 in colon cancer. DNA Cell Biol. 2007;26:311–20.PubMedCrossRefGoogle Scholar
  3. 3.
    Ventura A, Jacks T. MicroRNAs and cancer: short RNAs go a long way. Cell. 2009;136:586–91.PubMedCrossRefGoogle Scholar
  4. 4.
    Aqeilan RI, Calin GA, Croce CM. miR-15a and miR-16-1 in cancer: discovery, function and future perspectives. Cell Death Differ. 2010;17:215–20.PubMedCrossRefGoogle Scholar
  5. 5.
    Gartel AL, Kandel ES. miRNAs: little known mediators of oncogenesis. Semin Cancer Biol. 2008;18:103–10.PubMedCrossRefGoogle Scholar
  6. 6.
    Fujita S, Iba H. Putative promoter regions of miRNA genes involved in evolutionarily conserved regulatory systems among vertebrates. Bioinformatics. 2008;24:303–8.PubMedCrossRefGoogle Scholar
  7. 7.
    He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet. 2004;5:522–31.PubMedCrossRefGoogle Scholar
  8. 8.
    Koturbash I, Zemp FJ, Pogribny I, Kovalchuk O. Small molecules with big effects: the role of the microRNAome in cancer and carcinogenesis. Mutat Res. 2010;722:94–105.PubMedGoogle Scholar
  9. 9.
    Kloosterman WP, Plasterk RH. The diverse functions of microRNAs in animal development and disease. Dev Cell. 2006;11:441–50.PubMedCrossRefGoogle Scholar
  10. 10.
    Stefani G, Slack FJ. Small non-coding RNAs in animal development. Nat Rev Mol Cell Biol. 2008;9:219–30.PubMedCrossRefGoogle Scholar
  11. 11.
    Lu J, Qian J, Chen F, Tang X, Li C, Cardoso WV. Differential expression of components of the microRNA machinery during mouse organogenesis. Biochem Biophys Res Commun. 2005;334:319–23.PubMedCrossRefGoogle Scholar
  12. 12.
    Ng EK, Chong WW, Jin H, Lam EK, Shin VY, Yu J, et al. Differential expression of microRNAs in plasma of patients with colorectal cancer: a potential marker for colorectal cancer screening. Gut. 2009;58:1375–81.PubMedCrossRefGoogle Scholar
  13. 13.
    Calin GA, Ferracin M, Cimmino A, Di Leva G, Shimizu M, Wojcik SE, et al. A microRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med. 2005;353:1793–801.PubMedCrossRefGoogle Scholar
  14. 14.
    Schetter AJ, Leung SY, Sohn JJ, Zanetti KA, Bowman ED, Yanaihara N, et al. MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma. JAMA. 2008;299:425–36.PubMedCrossRefGoogle Scholar
  15. 15.
    Chim SS, Shing TK, Hung EC, Leung TY, Lau TK, Chiu RW, et al. Detection and characterization of placental microRNAs in maternal plasma. Clin Chem. 2008;54:482–90.PubMedCrossRefGoogle Scholar
  16. 16.
    Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA. 2008;105:10513–8.PubMedCrossRefGoogle Scholar
  17. 17.
    Park NJ, Zhou H, Elashoff D, Henson BS, Kastratovic DA, Abemayor E, et al. Salivary microRNA: discovery, characterization, and clinical utility for oral cancer detection. Clin Cancer Res. 2009;15:5473–7.PubMedCrossRefGoogle Scholar
  18. 18.
    Li J, Smyth P, Flavin R, Cahill S, Denning K, Aherne S, et al. Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells. BMC Biotechnol. 2007;7:36.Google Scholar
  19. 19.
    Nelson PT, Baldwin DA, Kloosterman WP, Kauppinen S, Plasterk RH, Mourelatos Z. RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain. RNA. 2006;12:187–91.PubMedCrossRefGoogle Scholar
  20. 20.
    Xi Y, Nakajima G, Gavin E, Morris CG, Kudo K, Hayashi K, et al. Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples. RNA. 2007;13:1668–74.PubMedCrossRefGoogle Scholar
  21. 21.
    El-Hefnawy T, Raja S, Kelly L, Bigbee WL, Kirkwood JM, Luketich JD, et al. Characterization of amplifiable, circulating RNA in plasma and its potential as a tool for cancer diagnostics. Clin Chem. 2004;50:564–73.PubMedCrossRefGoogle Scholar
  22. 22.
    Tsui NB, Ng EK, Lo YM. Stability of endogenous and added RNA in blood specimens, serum, and plasma. Clin Chem. 2002;48:1647–53.PubMedGoogle Scholar
  23. 23.
    Xie Y, Todd NW, Liu Z, Zhan M, Fang H, Peng H, et al. Altered miRNA expression in sputum for diagnosis of non-small cell lung cancer. Lung Cancer. 2009;67:170–6.PubMedCrossRefGoogle Scholar
  24. 24.
    Ahlquist DA, Skoletsky JE, Boynton KA, Harrington JJ, Mahoney DW, Pierceall WE, et al. Colorectal cancer screening by detection of altered human DNA in stool: feasibility of a multitarget assay panel. Gastroenterology. 2000;119:1219–27.PubMedCrossRefGoogle Scholar
  25. 25.
    Dong SM, Traverso G, Johnson C, Geng L, Favis R, Boynton K, et al. Detecting colorectal cancer in stool with the use of multiple genetic targets. J Natl Cancer Inst. 2001;93:858–65.PubMedCrossRefGoogle Scholar
  26. 26.
    Kalimutho M, Del Vecchio Blanco G, Gravina P, Cretella M, Mannucci L, Mannisi E, et al. Quantitative denaturing high performance liquid chromatography (Q-dHPLC) detection of APC long DNA in faeces from patients with colorectal cancer. Clin Chem Lab Med. 2010;48:1303–11.PubMedCrossRefGoogle Scholar
  27. 27.
    Kalimutho M, Del Vecchio Blanco G, Cretella M, Mannisi E, Sileri P, Formosa A, et al. A simplified, non-invasive fecal-based DNA integrity assay and iFOBT for colorectal cancer detection. Int J Colorectal Dis. 2011;26:583–92.PubMedCrossRefGoogle Scholar
  28. 28.
    Taylor CF, Field D, Sansone SA, Aerts J, Apweiler R, Ashburner M, et al. Promoting coherent minimum reporting guidelines for biological and biomedical investigations: the MIBBI project. Nat Biotechnol. 2008;26:889–96.PubMedCrossRefGoogle Scholar
  29. 29.
    Mestdagh P, Feys T, Bernard N, Guenther S, Chen C, Speleman F, et al. High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA. Nucleic Acids Res. 2008;36:e143.Google Scholar
  30. 30.
    Mestdagh P, Van Vlierberghe P, De Weer A, Muth D, Westermann F, Speleman F, et al. A novel and universal method for microRNA RT-qPCR data normalization. Genome Biol. 2009;10:R64.Google Scholar
  31. 31.
    Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:RESEARCH0034.Google Scholar
  32. 32.
    Ahmed FE, Jeffries CD, Vos PW, Flake G, Nuovo GJ, Sinar DR, et al. Diagnostic microRNA markers for screening sporadic human colon cancer and active ulcerative colitis in stool and tissue. Cancer Genomics Proteomics. 2009;6:281–95.PubMedGoogle Scholar
  33. 33.
    Koga Y, Yasunaga M, Takahashi A, Kuroda J, Moriya Y, Akasu T, et al. MicroRNA expression profiling of exfoliated colonocytes isolated from feces for colorectal cancer screening. Cancer Prev Res (Phila). 2010;3:1435–42.CrossRefGoogle Scholar
  34. 34.
    Link A, Balaguer F, Shen Y, Nagasaka T, Lozano JJ, Boland CR, et al. Fecal MicroRNAs as novel biomarkers for colon cancer screening. Cancer Epidemiol Biomarkers Prev. 2010;19:1766–74.PubMedCrossRefGoogle Scholar
  35. 35.
    Yu YJ, Majumdar AP, Nechvatal JM, Ram JL, Basson MD, Heilbrun LK, et al. Exfoliated cells in stool: a source for reverse transcription-PCR-based analysis of biomarkers of gastrointestinal cancer. Cancer Epidemiol Biomarkers Prev. 2008;17:455–8.PubMedCrossRefGoogle Scholar
  36. 36.
    Bartels CL, Tsongalis GJ. MicroRNAs: novel biomarkers for human cancer. Clin Chem. 2009;55:623–31.PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2011

Authors and Affiliations

  • Murugan Kalimutho
    • 1
    • 4
  • Giovanna Del Vecchio Blanco
    • 2
  • Serena Di Cecilia
    • 1
  • Pierpaolo Sileri
    • 2
    • 3
  • Micaela Cretella
    • 2
  • Francesco Pallone
    • 1
    • 2
  • Giorgio Federici
    • 1
  • Sergio Bernardini
    • 1
  1. 1.Department of Internal MedicineUniversity of Rome “Tor Vergata”RomeItaly
  2. 2.Gastroenterology UnitUniversity Hospital Tor Vergata, University of Rome Tor VergataRomeItaly
  3. 3.Surgical UnitUniversity Hospital Tor Vergata, University of Rome Tor VergataRomeItaly
  4. 4.Centre for Cancer Research and Cell Biology (CCRCB)Queen’s UniversityBelfastNorthern Ireland, UK

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